The Molecular Pathobiology Section's functional objectives are to investigate the genetic molecular structure of pathogens, to define the role of gene products in pathogenic mechanisms and to perform studies directed toward development of vaccines using molecular or synthetic production of immunogenic peptides from microbial agents. Our major emphasis is focused on cloning and expression of genes relevant to the toxic components of Bordetella pertussis i.e., the bacteria responsible for whooping cough. Our immediate goal is to detoxify pertussis by molecular manipulation of the genome, thus producing a "cleaner bug" for use in second generation vaccine development. Using monoclonal antibodies, we are currently working on the expression of pertussis toxin i.e., lymphocytosis promoting factor, which we have cloned and sequenced. Our long term interests are in the identification and understanding of epitopes which stimulate synthesis of protective antibodies and the development of third generation vaccines such as protein subunits and synthetic peptide antigens. Development of a safer "new generation" pertussis component vaccine using molecular cloning of Bordetella pertussis genes has been hampered by the inherent difficulties in identifying DNA fragments containing specific pertussis genes for protective antigens. Cloning strategies which rely expression of protein in E. coli have not been very successful probably because of differences in the gene regulation transcriptional signals. Alternate more direct strategies using oligonucleotide probes coding for specific portions of proteins may be more successful. The polycistornic nature of prokaryotes suggest that the genes coding for the protein subunits of pertussis toxin may be in a tandem arrangement regulated by one operator. Recently, we have cloned and partially sequenced a 4.2 kb EcoR1/BamH1 DNA fragment containing at least two of the five subunit genes of pertussis toxin.